1. Technical Field of the Invention
The present invention is directed to identification of the source of biologic specimens such as medicinal materials, particularly to a method of determining the identity of plants used in the medicinal materials and a kit used for the identification. The invention is exemplified by methods and kits for differential identification of materials derived from one of three different species of Ilex. 
2. Description of the Related Art
An effective method of authentication of traditional Chinese medicinal materials is necessary for the development of the industry, as it provides the necessary protection for consumers, minimizes unfair business competition, and prevents health hazards due to materials that adulterate the medicinal materials. Traditionally, the authentication of Chinese herbs has relied upon morphological and histological inspection. For many biologic materials, this method is unreliable.
Eukaryotic genes for ribosomal RNA (rDNA) are normally clustered in an array of multiple tandemly repeated copies of the cistron of 18S-ITS1-5.8S-ITS2-28S (Hillis, and Dixon, 1991, The Quarterly Review of Biology, 66: 411-453). The sequence that separates the 18S and 5.8S rRNA genes is designated as ITS1 (Internal Transcribed Spacer 1) and the sequence between 5.8S and 28S is designated as ITS2. While the coding regions of the rDNA genes are highly similar, the sequence conservation within the ITS1 and ITS2 regions is much lower. Nevertheless, within a given individual organism or species, the sequences of rDNA ITS1 and ITS2 are usually similar as a result of gene conversion and crossing over. Methods have been developed based on the sequence polymorphism of rDNA ITS1 and ITS2 regions, such as the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique. However, conventional methods of authenticating traditional Chinese medicinal materials are limited because they rely on the presence of suitable restriction enzyme cutting sites in the amplified DNA sequence. In the absence of an expected restriction enzyme cutting site, which may result from a sequence polymorphism, definitive authentication of a specimen is not possible. Another approach to determine a species origin for a biologic material has been the use of short specific probes for DNA hybridization assays. The success of this method is dependent on the identification of highly specific target sequences. When one highly specific target sequence cannot be identified, a combination of short hybridization probes can be used. However, this will increase the likelihood of obtaining false positive results. In addition, the low hybridization signals generated with hybridization assays often introduce another source of ambiguity in interpretation of the test result. Therefore, there is a need for a reliable method of authentication of biologic materials.
U.S. Pat. Nos. 5,876,977 and 6,309,840 disclose a PCR-RFLP based method for authenticating some species of plants used in Chinese medicinal materials based on differences in ribosomal RNA gene sequences.